Ketamine ameliorates activity-based anorexia of adolescent female mice through changes in the prevalence of NR2B-containing NMDA receptors at excitatory synapses that are in opposite directions for of pyramidal neurons versus GABA interneurons In medial prefrontal cortex

A previous study showed that a single sub-anesthetic dose of ketamine (30 mg/kg-KET, IP) has an immediate and long-lasting (>20 days) effect of reducing maladaptive behaviors associated with activity-based anorexia (ABA) among adolescent female mice. This study sought to determine whether synaptic plasticity involving NR2B-containing NMDA receptors (NR2B) at excitatory synapses in the prelimbic region of medial prefrontal cortex (mPFC) contributes to this ameliorative effect. To this end, quantitative electron microscopic analyses of NR2B-subunit immunoreactivity at excitatory synapses of pyramidal neurons (PN) and GABAergic interneurons (GABA-IN) were conducted upon layer 1 of mPFC of the above-described mice that received a single efficacious 30 mg/kg-KET (N=8) versus an inefficacious 3 mg/kg-KET (N=8) dose during the food-restricted day of the first ABA induction (ABA1). Brain tissue was collected after these animals underwent recovery from ABA1, then of recovery from a second ABA induction (ABA2), 22 days after the ketamine injection. For all three parameters used to quantify ABA resilience (increased food consumption, reduced wheel running, body weight gain), 30 mg/kg-KET evoked synaptic plasticity in opposite directions for PN and GABA-IN, with changes at excitatory synapses on GABA-IN dominating the adaptive behaviors more than on PN. The synaptic changes were in directions consistent with changes in the excitatory outflow from mPFC that weaken food consumption-suppression, strengthen wheel running suppression and enhance food consumption. We hypothesize that 30 mg/kg-KET promotes these long-lasting changes in the excitatory outflow from mPFC after acutely blocking the hunger and wheel-access activated synaptic circuits underlying maladaptive behaviors during ABA.


Introduction
Anorexia nervosa (AN) is characterized by self-starvation, heightened anxiety and excessive exercising (Carrera et al. 2012;Crisp 1967; Kron et al. 1978), culminating in severe body weight loss (Kaye et al. behaviors. Indeed, a single intraperitoneal (IP) injection of ketamine did ameliorate all of the above-listed core symptoms in mice 14-22 days post-injection (Chen et al. 2018), while we also noted individual differences in vulnerability to anorexia-like behaviors and of their responsiveness to the ketamine treatment. Results from this ABA study, as well as the well-recognized e cacy of low doses of ketamine as an antidepressant (Zorumski et al. 2016), prompted clinicians to test the e cacy of ketamine in treating AN. These studies also yielded mixed results, with some but not all patients experiencing clear remission of disordered eating symptoms (Calabrese 2022;Scolnick et al. 2020; Schwartz et al. 2021).
Thus, the aim of this study was to determine whether changes in the expression pattern of NR2B at excitatory synapses might be associated with individual differences in vulnerability to ABA and of their responsiveness to ketamine.
Although multiple brain regions are likely to become engaged in ABA's maladaptive behaviors and responses to ketamine, this study focused on a single brain region: medial prefrontal cortex (mPFC). The rationale for studying the mPFC is that this brain region has been implicated heavily in mental illnesses like AN that stem from anxiety. Compared to controls, the PFC of patients diagnosed with AN exhibit diminished activities at rest and abnormally increased activities when presented with food images (Uher et al. 2004). Using single photon emission computed tomography (SPECT), hypoperfusion of mPFC at normal resting conditions was found for subjects diagnosed with AN, relative to healthy controls (Takano et al. 2001). Moreover, maladaptive food choices by subjects diagnosed with AN is linked to stronger PFC-striatum connectivity (Foerde et al. 2015). In mice, suppression of hyperactivity evoked by food restriction under the ABA model correlates with enhanced synaptic contacts formed by glutamic acid decarboxylase (GAD)-positive GABAergic axon terminals onto cell bodies of pyramidal cells in layer 5 of the mPFC (Chen et al. 2017b; Chen et al. 2016). Chemogenetic activation 3 hours prior to food availability of mPFC pyramidal cells that project to the dorsal medial striatum (mPFC◊DMS) increases food restriction-evoked wheel running, referred to as food-anticipatory activity (FAA) (Gallardo et al. 2014) signi cantly, while suppression of these cells at the same hour of the day reduces FAA (Santiago et al. 2021). In contrast to the chemogenic modulatability of FAA, chemogenetic modulation of the food consumption-promoting pathway was not possible (Du et al. 2022). Speci cally, activation of mPFC pyramidal neurons projecting to the dorsal raphe nucleus (mPFC◊DR) (Jankowski and Sesack 2004) is known to increase feeding behaviors via activation of GABAergic neurons in DR to which these pyramidal neurons project (Nectow et al. 2017) but chemogenetic activation of the mPFC◊DR pathway of ABA animals could not induce feeding: we learned that this was due to the overriding strong inhibition of this pathway by GABAergic neurons in the mPFC (Du et al. 2022).
In this way, data from rodent and human studies point to the mPFC as a key region for an individual's decision to forage (run) or eat when experiencing starvation. Besides these accumulating evidence regarding the central role of mPFC in AN and ABA's maladaptive behaviors, a study investigating ketamine's mechanism as an antidepressant indicates that functional connectivity between the mPFC and ventral hippocampus is necessary for ketamine's action (Carreno et al. 2016). These ndings led us to focus our analysis on the mPFC for detecting changes in synaptic circuitry in response to the experience of ABA following treatments with e cacious versus ine cacious doses of ketamine. Might mPFC show similar positive correlation between the level of NR2B subunits and vulnerability to ABA, as was observed for the hippocampus (Chen et al. 2017a)? This seemed possible, since activities between the hippocampus and mPFC become synchronized when mice are exposed to anxiogenic environments (Adhikari et al. 2010), such as starvation, and mPFC receives inputs from the ventral hippocampus (Adhikari et al. 2011). Another reason for focusing on NR2B was that ketamine's antidepressant action is occluded by genetic deletion of NR2B-subunits in pyramidal neurons (Miller et al. 2014;Miller et al. 2017) or in GABA-interneurons (GABA-IN) (Gerhard et al. 2020;Gerhard et al. 2016) and NR2B-containing NMDARs in prefrontal cortex have been shown to be important for contextual memory formation (Zhao et al. 2005). Prompted by these ndings, we sought to assess whether the redistribution of NR2B at excitatory synapses formed upon pyramidal neurons versus GABA-IN might be evoked by ketamine and be related to ketamine's e cacy of reducing ABA's maladaptive behaviors. Finally, we focused our analysis upon layer 1 of mPFC. This choice was based on earlier works indicating that changes in dendritic spine sizes and numbers could be detected within apical tufts of mPFC following ketamine treatment (Deyama et

Methods
Animals FR started at 1pm on P41 for ABA1 and on P55 for ABA2. The 2-hour food access period of each FR day started at 7pm and ended at 9pm. Lights were turned on from 7am to 7pm everyday (light phase). Lights were off from 7pm to 7am (dark phase). 3mg/kg or 30mg/kg ketamine were injected at 6pm of ABA1 FR2, 1 hr before feeding. FAA (food anticipatory activity) period refers to the hours of 1 pm to 7 pm, when hunger evokes hyperactivity of ABA animals.
The Activity-based anorexia (ABA) procedure Figure 1 shows the timeline of ABA. ABA1 started on postnatal day 36 (P36) for all animals. On this day, group-housed animals began to be singly housed. During P36 to P40, animals underwent acclimation to the addition of a wheel in its cage, and were exposure to wet food (Clear H 2 O brand DietGel 76A in plastic cups, 0.998 kcal/g, 4.7% protein, 17.9% carbohydrates, 1.5% fat, 73.4% moisture), in addition to dry food pellets that they had received before weaning (LabDiet PMI Nutrition Int'l, Brentwood, MO's #5001; 10% fat, 20% protein, 70% carbohydrate, 4.07 gross energy kcal/g, 3.02 metabolizable kcal/g; alternatively LabDiet Rodent Diet 20 EXT (5053); 20% protein, 4.5% crude fat, 6% crude ber, 7% ash, 12% maximum moisture with 4.07 gross energy kcal/g, equal to 3.07 metabolizable energy kcal/g). This period was used for acquiring baseline wheel running, body weight and food consumption data. These data were collected daily, to the end of ABA2, for monitoring changes in wheel running, body weight and food consumption during different periods of the experiment.
Food restriction (FR) of ABA1 lasted for three days, beginning at 1 pm on P41 (FR1) until 1 pm on P44 (FR3), during which time animals' food access was limited to 2 hours, from 7 pm to 9 pm. During the hours without food, a water gel cup (Clear H 2 O brand Hydrogels Produce #70-01-5022, 0 kcal/g) was placed where the wet food had been placed during acclimation, and the dry food hopper remained in place but was empty. Thus, food restriction began on P41 by removing both wet and dry food at 1 pm. Food was given at 7 pm, then taken away at 9 pm until 7 pm of the next day. Animals tended to eat less food on the rst day of FR (FR1) than on other FR days.
Ketamine administration occurred on P42. On this day, animals were randomly assigned to receive either 3 mg/kg or 30 mg/kg of ketamine. Ketamine was diluted with sterile saline, yielding a volume of 0.2 cc/20 g of body weight) and injected intraperitoneally (i.p.) at 6 pm, one hour before food access that began at 7pm.
Recovery period lasted from P44 to P51. During this period, wheels were taken away and food access returned to being ad libitum. Speci c days during recovery are referred to in the Results section as R2, R6, etc., referring to the 2nd, 6th day, etc., of recovery, relative to the end of the FR period.
Re-acclimation to the wheel for ABA2 began on P51 and continued for 4 days, until P55. The extent of wheel running and food consumption during the re-acclimation period was used to determine the new baseline for the second FR period of ABA2. The second FR period of ABA2 lasted for four days, from P55 (FR1) to P59 (FR4), during which time relapse of anorexia-like behaviors of excessive exercise, voluntary food restriction and body weight loss were assessed. Finally, starting on P59, all animals underwent a second recovery for 5 days, during which time wheels were removed and food access became ad libitum.
The body weights of all animals were measured daily at around 1pm for non-FR days and at 7pm and 9pm for FR days. The weights of dry and wet food consumption were also measured whenever body weights were measured, then converted to total kilocalories (kCal) consumed per day. Wheel activity was recorded manually whenever body weights were measured. Wheel activity was also recorded at oneminute resolution during all hours of wheel accessibility. For both FR periods, animals were moved to a new cage right after the end of FR. Though all mice were females, the estrous cycle was not monitored because 1. puberty was too immature for a stable estrous cycle, 2. FR disrupts the cycle regardless, and 3. vaginal smears, required to obtain data for estrous cycle, exacerbates anxiety of animals already stressed by FR and considered undesirable perturbations (Chen et al. 2016).
All aspects of animal handling for ABA were performed by Dr. Yi-Wen Chen. Data on body weight, food consumption and wheel counts of these ketamine-injected animals have been published (Chen et al. 2018).

Elevated Plus Maze
The Elevated Plus Maze (EPM) was used to test for anxiety-like behaviors. This was performed on P49 and P62 during the dark cycle for 10 minutes for each animal. This part of the study using the EPM to measure anxiety-like behaviors of mice was conducted by Dr. Yi-wen Chen and is already published (Chen et al. 2018). These EPM data are not presented in the current study.

Histological Preparation
On the morning of P63 (± 1 day), all 16 animals were anesthetized with urethane (1000-1500 mg/kg) by IP injection, followed by transcardial perfusion with 50ml of phosphate-buffered saline (PBS; 0.01 M phosphate buffer, 0.9% sodium chloride, pH 7.4) containing Heparin (20 U/ml). This was followed immediately by perfusion with 0.1M phosphate buffer (pH 7.4) containing 4% paraformaldehyde (PFA, EM Sciences, Hat eld, PA, USA). Glutaraldehyde was not included in the perfusate, to minimize loss of antigenicity of brain proteins. Brains were stored in 4% PFA in 0.1M phosphate buffer at 4 o C until use, 2 years later.

Immunocytochemistry
The vibratome tissue underwent a pre-embed immunocytochemical procedure to immunolabel for a postsynaptic protein, drebrin A. Immunocytochemical procedures and the electron microscopic data pertaining to drebrin A localization have been published elsewhere (Temizer et al., 2022), and thus, are not presented here. NR2B-subunits of NMDA receptors (NR2B) were detected within these sections by the post-embed immunogold method, after post-xing vibratome sections in 2% glutaraldehyde in PBS for 30 min at room temperature and the osmium-free procedure for ultrastructural preservation (Aoki et al. 2000;Phend et al. 1995;Kharazia et al. 1996), as detailed below.
Osmium-free tissue processing All steps of this process were performed on ice. Vibratome-cut sections were washed in maleate buffer, pH 6.0 (MB) 2 times for 5 minutes each. Sections underwent the following steps to enhance ultrastructural preservation and contrast: immersion in 1% tannic acid in MB for 40 minutes, followed by two rinses using MB, then 1% ltered uranyl acetate in MB for 40 minutes, followed by two rinses in MB, then 0.2% iridium tetrabromide in MB for 20 minutes, followed by two rinses using MB, then incubation in 50% ethanol for 5 minutes, 70% ethanol for 3 minutes, then in 1% para-phenylenediamine hydrochloric acid in fresh 70% ethanol for 15 minutes while protected from light, followed by rinses using 70% ethanol.
Tissues were stored overnight at 4 o C in 1% ltered uranyl acetate in 70% ethanol, shielded from light, within scintillation vials.
On the following day, sections were prepared for in ltration with the embedding matrix by dehydrating them in progressively higher concentrations of ethanol, up to 100%, followed by immersion in 100% acetone three times for 5 minutes each, 1:1 volume of EMBED812 (EMSciences Catalog #14900) and 100% acetone for 3 hours at room temperature and 1 hour at 50 o C. Next, sections were in ltrated in 100% EMBED812 for 4 hours with 1 of the hours at 50 o C, then at-embedded between two sheets of Aclar plastics. Flat-embedded tissues were cured at 60 o C for 24 to 36 hours, with weights added on the Aclar sheets to induce atness of the sections. After re-embedding the sections in Beem capsules, ultrathin sections were prepared using a diamond knife, then collected onto formvar-coated EM grids (400 mesh, nickel thin bars, EMSciences). These ultrathin sections were processed for post-embedded gold (PEG) immunolabeling.
Primary and secondary antibodies for NR2B immunocytochemistry by the PEG procedure The primary antibody for the NR2B subunit of NMDAR (GRIN2B gene product) was a rabbit polyclonal antibody (Millipore Corp, Polyclonal Rb Anti-NR2B 06-600, Lot #251403), applied using previously published procedures Aoki et al. 2005). This antibody identi es the C-terminal amino acids 1437-1456 (KFNGSSNGHVYEKLSSIESDV). Speci city of the antibody was shown based on recognition of a single band from rat brain microsomal preparation of molecular weight ~ 180kD by Western blotting that excludes immunoreactivity to the NR1 subunit or NR2A subunit of NMDARs (Rinaldi et al. 2007). The antigen recognized by this antibody is expressed with a distinct developmental (Corson et al. 2009;Sheng et al. 1994) and subcellular (Aoki et al. 2005; Fujisawa and Aoki 2003) distribution pattern, compared to the NR2A subunit. The secondary antibody for NR2B was goat anti-rabbit IgG conjugated to 10 nm colloidal gold particles, purchased from Electron Microscopy Science (Cat. #25109).
The post-embed gold procedure (PEG) for detecting NR2Bcontaining NMDARs (NR2B) The PEG procedure was used to label NR2B by applying primary and secondary antibodies directly onto ultrathin sections collected on formvar-coated EM grids, as detailed in previous publications from this lab prior to incubation in the solutions used for the PEG procedure. This timing between the start of the PEG procedure and ultrathin sectioning was important for ensuring that the sections remained adhered securely to formvar coated-grids while also avoiding waning of antigenicity due to air exposure. Fifty-µl of a 0.1M Tris-buffered saline with 0.1% Triton X-100 (TBST)-pH 7.4 droplet was put into each division of a silicon mat. There were 16 ultrathin sections processed in parallel, derived from 16 different animals. All grids were inserted under a droplet of TBST-pH 7.4 with the ultrathin section facing upwards with respect to the droplet. Next, the sections were transferred in the same manner into a droplet of 50 µl of the primary antibody for NR2B diluted in TBST-pH 7.4 at a dilution of 1:100 (10 µg/ml). Sections were incubated in the primary antibody solution overnight at room temperature within a humidi ed chamber. On the following day, sections were rinsed three times at 10-minute intervals with 50 µl of TBST-pH 7.4 droplets in the same manner, followed by incubation in a droplet of TBST-pH 8.4. Then the tissues were incubated in the secondary antibody conjugated to 10 nm colloidal gold particles, diluted in TBST-pH 8.4 (1:40). After an hour of incubation at room temperature within a humidi ed chamber, sections were rinsed with TBST-pH 8.4 three times and 10 minutes each, and then in deionized water three times at 10-minute intervals. Tissues were xed in 1% glutaraldehyde diluted in deionized water for 10 minutes, rinsed in deionized water, and air-dried.
Ultrathin sections designated to serve as immunocytochemical controls were collected on formvar-coated grids, strictly in parallel with those sections described above and were processed for PEG labeling in parallel, except that the step for incubating with the primary antibody was substituted by a step of incubating in TBST-pH 7.4 that lacked the primary antibody.

Counterstaining
Counterstaining was done in order to increase the contrast of tissues, to facilitate interpretation of the ultrastructure. Lead citrate was prepared as described by Reynolds (Reynolds 1963). A few droplets of lead citrate, using a syringe and lter, were put on a piece of dental wax plate within a petri dish lined with sodium hydroxide pellets. Each EM grid with a tissue on it was immersed in a new droplet of lead citrate for 20 seconds. Immediately after, the tissues on the grid were washed in four beakers of deionized water, then dried on a piece of lter paper. Grids were air-dried for a minimum of 2 hours before viewing under the EM or placed in grid storage containers.

Electron Microscopy
Layer 1 of the prelimbic area of mPFC was identi ed by the absence of pyramidal cells and presence of pia mater. All images were magni ed to 40,000x, within 5 µm from cortical surface, then digitally captured using a Hamamatsu CCD camera, attached to a JEOL 1200XL electron microscope, developed by AMT (Boston, MA). Images were taken and analyzed systematically and strictly in the order of encounter, without knowledge of the animal's behavioral characteristics. Images of 200 synapses (± 5%) were captured from each animal's mPFC. Excitatory synapses were identi ed based on the presence of thick postsynaptic densities (PSD) and clusters of vesicles approximately 50 nm in diameter in the opposing presynaptic axon terminal (Peters et al. 1991). The postsynaptic element was determined to be spines, based on the absence of mitochondria and microtubules (Peters et al. 1991). Excitatory synapses on dendritic shafts of GABAergic interneurons were identi ed by the presence of mitochondria and microtubules and the existence of thick PSDs opposite to axon terminals lled with vesicles (White and Keller 1989). Inhibitory synapses were identi ed by the cluster of vesicles within the presynaptic terminal and absence of thick PSDs. Images were analyzed and annotated on ImageJ bundled with Java 1.8.0_172. Figures containing electron micrographs were prepared using Adobe Photoshop 2023 version.
Quanti cation of the ultrastructural localization of NR2B NR2B locations relative to excitatory synapses were quanti ed using digitized electron micrographs, offline, guided by the ultrastructural categorization of subcellular locations as shown in Fig. 3a. Excitatory synapses occurred at heads of dendritic spines, presumed to belong to pyramidal neurons. Excitatory synapses occurring along dendritic shafts were presumed to belong to GABAergic interneurons (GABA-IN) (White and Keller 1989).

Figures 3b and 3c
show examples of PEG immunolabels for NR2B. PEG labels were identi ed by de ned circular black dots that were 10 nm in size, as expected. In both panels of Fig. 3 and for every excitatory synapse identi ed in this study, PSD was detectable as a thick dark band along the intracellular surface of the plasma membrane opposed to the plasma membrane of vesicle-containing axon terminals.
NR2B immunolabel appeared on the extrasynaptic plasma membrane of both pre-and postsynaptic sides, in the cytoplasm of both pre-and postsynaptic sides, near (~ 10 nm from) the lipid bilayer of plasma membrane and at the synaptic plasma membrane (< 10 nm from the lipid bilayer of the synaptic plasma membrane) on the presynaptic sides, near (~ 10 nm) and over (< 10 nm) the PSD of the postsynaptic side. They also appeared in the synaptic cleft, i.e., within the extracellular space in between the presynaptic and postsynaptic membranes of a synapse. This extracellular cleft location has been reported earlier for glutamate receptors detected by the PEG procedure, presumed to re ect the displacement of colloidal gold from the antigenic site due to the bridging of the primary and secondary antibody IgG molecules (ca. 15 nm in length, each) and of colloidal gold that are 10 nm in diameter (Valtschanoff & Weinberg, 2001). PEG particles associated with mitochondria were regarded as nonspeci c labeling and excluded from counting in the cytoplasm.
NR2B levels of each animal were quanti ed in two ways: (1) as the proportion of all synapses encountered that were immunolabeled by at least one PEG particle at each of the subcellular locations; (2) as the summed total counts of PEG particles at each of the subcellular locations, per the total number of synapses encountered. Excitatory synapses were considered immunolabeled if they were associated with one or more PEG particle. Using this criterion, analysis of over 200 excitatory synapses from control ultrasections that were incubated with TBST lacking the primary antibody resulted in zero immunolabeling, since none were associated with PEG particles.

Statistical Analysis
Data was processed using an original algorithm created on Python Language Reference, version 3.6.9, on Google Colab. Pearson's correlation analysis was performed on Graphpad Prism Version 7.01 to identify correlations between body weight, food consumption, wheel counts and prevalence of NR2B at excitatory synapses. Statistical signi cance was determined by p < 0.05 and R < -0.7 or R > 0.7, and found using another original program created on Python Language Reference, version 3.6.9, on Google Colab. Two sample mean t-tests were performed to analyze group behavioral differences, where p < 0.05 was considered as statistically signi cant, using GraphPad Prism Version 7.01. Graphs were prepared using GraphPad Prism.

Results
The behavioral data of the animals whose brains were anatomically analyzed in this study have already been published (Chen et al. 2018). These behavioral data will be presented here as correlations to the anatomical ndings from the current study. The current study examined correlations between three ante mortem values -wheel running, food consumption and body weight -and the levels and locations of NR2B in relation to excitatory synapses in layer 1 of the prelimbic area of mPFC. NR2B was labeled by the PEG method.

Areal Density of Excitatory Synapses
We compared the areal density of excitatory synapses belonging to pyramidal neurons and GABA-IN in layer 1 (mostly layer 1a, within 50 µm from pial surface) of the mPFC. Layer 1 of the 30 mg/kg cohort exhibited a signi cantly lower density of axo-spinous excitatory synapses (presumably of pyramidal neurons (White and Keller 1989)) than the 3 mg/kg cohort (Fig. 4a, mean ± SEM: 0.3700 ± 0.0143 for the 30 mg/kg cohort, 0.4656 ± 0.0170 for the 3 mg/kg cohort, p = 0.0007). The probability of encounter with randomly positioned objects would be lower for smaller objects (Sterio 1984). However, there was no signi cant difference in the average spine area between the two cohorts, calculated by averaging 50 spine areas of each animal (mean ± SEM in units of synapses per µm 2 : 226.2 ± 58.0 for the 30 mg/kg cohort; 341.1 ± 86.3, for the 3 mg/kg cohort, p = 0.484). This suggests that the lower areal density of axospinous excitatory synapses seen in the 30 mg/kg cohort did not result from decrease of spine sizes.

The Proportion of Excitatory Synapses Immunolabeled for NR2B Postsynaptically
Control ultrathin sections that were incubated in buffer lacking the primary antibody showed no PEG labeling at or near any of the 200 excitatory synapses that were sampled. This observation indicated that excitatory synapses with one or more PEG particles could be considered immunolabeled.
The proportion of excitatory synapses immunolabeled by NR2B was not different across cohorts, based on tallies of PEG particles across all synaptic locations -namely at and near the PSD, on extrasynaptic portions of the plasma membrane, or in the synaptic cleft or cytoplasm of spines forming excitatory synapses (p = 0.0956; mean ± SEM of 0.5968 ± 0.0643 for the 30 mg/kg cohort; 0.4553 ± 0.04619 for the 3 mg/kg cohort) or at excitatory synapses on dendritic shafts of GABA-IN (p = 0.0643; 0.5968 ± 0.0643 for the 30 mg/kg cohort; 0.4553 ± 0.04619 for the 3 mg/kg cohort ). Also, no group difference was found, when the PEG particles were tallied for membranous locations (at the extrasynaptic membrane, at or near the PSD). However, closer inspection revealed that the 30 mg/kg cohort exhibited a signi cantly higher proportion of spines immunolabeled for NR2B at the cleft and in the cytoplasm of spines (Fig. 5b, mean ± SEM at spines' excitatory synaptic clefts: 0.0485 ± 0.0088 for the 30 mg/kg cohort, 0.0194 ± 0.0047 for the 3 mg/kg cohort, p = 0.0114; Fig. 5c, mean ± SEM of spine heads with PEG particles in cytoplasm: 0.254 ± 0.028 for the 30 mg/kg cohort, 0.186 ± 0.012 for the 3 mg/kg cohort, p = 0.0410).
At excitatory synapses of dendritic shafts of GABA-IN, no differences in the proportion immunolabeled for postsynaptic NR2B were found between the two cohorts at synaptic clefts (Fig. 5e, mean ± SEM: 0.0450 ± 0.0109 for the 30 mg/kg cohort, 0.0349 ± 0.0136 for the 3 mg/kg cohort, p = 0.571), in the cytoplasm near excitatory synapses (Fig. 5f, mean ± SEM: 0.432 ± 0.0569 for the 30 mg/kg cohort, 0.367 ± 0.0370 for the 3 mg/kg cohort, p = 0.353) or on the plasma membrane of the pre-or postsynaptic sides (not shown).
The level, rather than the proportion, of NR2B immunolabeled per excitatory synapse on dendritic spines and dendritic shafts yielded similar results. Speci cally, the level of NR2B labeled at synapses of dendritic spines were signi cantly greater for the 30 mg/kg cohort, compared to the 3 mg/kg cohort at synaptic clefts (mean ± SEM: 0.0508 ± 0.0097 for the 30 mg/kg cohort, 0.0202 ± 0.0053 for the 3 mg/kg cohort, p = 0.0149) and in the cytoplasm (mean ± SEM: 0.410 ± 0.064 for the 30 mg/kg cohort, 0.261 ± 0.021 for the 3 mg/kg cohort, p = 0.0425) but not at the postsynaptic plasma membranes. There were no differences in the level of NR2B levels between the two cohorts at clefts of excitatory synapses on dendritic shafts (mean ± SEM: 0.0507 ± 0.012 for the 30 mg/kg cohort, 0.0395 ± 0.018 for the 3 mg/kg cohort, p = 0.610), in cytoplasm (0.916 ± 0.22 for the 30 mg/kg cohort, 0.534 ± 0.065 for the 3 mg/kg cohort, p = 0.122) or elsewhere, either.

Correlational Analyses of the Proportion of Excitatory Synapses Immunolabeled for NR2B with Food Consumption
Within the mPFC, the activation of pyramidal neurons projecting to dorsal raphe (DR) have been shown to promote food consumption of animals undergoing ABA, while GABAergic inhibition of this population of mPFC pyramidal neurons has been shown to underlie suppression of food consumption (Du et al. 2022). This nding prompted us to analyze the possible link between ketamine-mediated increase in food consumption of animals that had experienced ABA and the excitatory synapses formed on pyramidal neurons and GABA-IN of the mPFC. Compared to the control group with vehicle injection (not included in this anatomical analysis), the two ketamine cohorts ate signi cantly more during the food-restricted period of ABA1, FR2, corresponding to the day that ketamine was injected. Additionally, the 30 mg/kg cohort, but not the 3 mg/kg cohort also ate more on the day following the single ketamine injection, namely FR3 of ABA1. During ABA2, 14 days after ketamine injection, only the 30 mg/kg ketamine cohort ate signi cantly more than the control group during the food-restricted period of ABA2, spanning FR2 through FR4 (Chen et al. 2018). Prompted by these differences in food consumption behaviors across the two ketamine cohorts, correlation analyses between food consumption and the proportion of dendritic spines and shafts immunolabeled for postsynaptic NR2B at excitatory synapses during ABA1 FR, ABA2 FR, and recovery phases were conducted to determine whether individual differences in NR2B expression at excitatory synapses could have contributed to individual differences in the responsiveness to ketamine in terms of food consumption.
Correlations at axo-spinous excitatory synapses of pyramidal neurons. Fig. 6 shows the R-values of correlations between the proportion of excitatory synapses with postsynaptic NR2B levels and food consumption recorded for each of the days spanning ABA1 and ABA2. There were clear differences in the patterns between the two ketamine cohorts ( Fig. 6a and b). The two time points that showed strong divergence between the cohorts were ABA2 R3 and ABA1 R4, which were examined more closely (Fig. 7).
Closer inspection of ABA2 R3 revealed that the 30 mg/kg cohort showed signi cantly higher food consumption than the 3 mg/kg cohort (mean ± SEM in units of kcal: 21.9 ± 0.894 for the 30 mg/kg cohort, 15.8 ± 0.962 for the 3 mg/kg cohort, p = 0.0005 by unpaired t-test, Fig. 7c). The 30 mg/kg cohort showed no correlation between NR2B immunoreactivity at dendritic spines and food consumption on any single day of recovery or average consumption across the days of recovery. In sharp contrast to the 30 mg/kg cohort, the 3 mg/kg cohort showed a signi cantly negative correlation between food consumption and the proportion of spines immunolabeled for postsynaptic NR2B at all locations except for near PSD (Pearson's R = 0.2117, p = 0.6486 for the 30 mg/kg cohort; Pearson's R=-0.7892, p = 0.020 for the 3 mg/kg cohort, Fig. 7d). The negative correlation indicates that those individuals consuming the most were those that expressed NR2B minimally at excitatory synapses on pyramidal neurons. These ndings indicate, together, that enhanced excitability of pyramidal neurons dictated suppression of food consumption of the 3 mg/kg cohort. Conversely, the 30 mg/kg cohort was likely to have enhanced food consumption through weakening of food-suppressing excitatory pathway(s) that remained active in the brains of the 3 mg/kg cohort (discussion expanded with accompanying gure in the Discussion section).
By comparison, for both cohorts, correlations between food consumption and NR2B-immunoreactivity at dendritic spines were much weaker during recovery from ABA1 (Fig. 7b). For example, on ABA1 R4, when the 30 mg/kg cohort had signi cantly higher food consumption than the 3 mg/kg cohort (mean ± SEM in units of kcal: 20.3 ± 1.50 for the 30 mg/kg cohort, 13.81 ± 0.87 or the 3 mg/kg cohort, p = 0.002 by unpaired t-test, Fig. 7a (Fig. 7e) and ABA2 (Fig. 7f) and only for the 30 mg/kg cohort. Speci cally, on ABA1 R6 and ABA2 R2, a signi cantly positive correlation was found in the 30mg/kg cohort between food consumption and the proportion dendritic shafts immunolabeled for postsynaptic NR2B at the cleft, at the PSD, and near the PSD (Pearson's R = 0.8058, p = 0.0288 for the 30 mg/kg cohort on ABA1 R6, Fig. 7e; Pearson's R = 0.8231, p = 0.0229 for the 30 mg/kg cohort on ABA2 R2, Fig. 7f), indicating that those individuals with the greatest capacity to consume food expressed NR2B at excitatory synapses on GABA-IN most robustly, presumably suppressing the mPFC's excitatory out ows that mediate suppression of food consumption.
In contrast, for the 3 mg/kg cohort, only a weakly positive correlation trend was found on ABA1 R6 (Pearson's R = 0.5888, p = 0.1246 for the 3 mg/kg cohort, Fig. 7e), and no trend was found on ABA2 R2 (Pearson's R = 0.3901, p = 0.3394 for the 3 mg/kg cohort, Fig. 7f). This lack of trend for the 3 mg/kg cohort supports the interpretation that the ine cacious dose could not trigger the increased expression of NR2B at excitatory synapses of GABA-IN for suppressing the food consumption-suppressive excitatory out ow. The appearance of correlations for the NR2B-immunoreactivity during ABA1 for axo-shaft excitatory synapses of GABA-IN and not for the excitatory synapses of pyramidal neurons' dendritic spines indicates that excitatory synapses on GABA-IN responded more quickly to the 30 mg/kg ketamine injection than the pyramidal neurons.
All data reported above were analyzed based on values of the proportion of dendritic spines or shafts immunolabeled for NR2B at excitatory synapses. Correlation analyses performed by counting every single NR2B immunolabel and normalizing the sum by the number of axo-spinous or axo-shaft excitatory synapses (i.e., NR2B levels) also yielded signi cance for the same days of food consumption.
Together, these data indicate that the long-lasting gain of ABA resilience by the 30 mg/kg dose, measured as an increase of food consumption more than 20 days after the single injection, correlated with increases of NR2B-immunoreactivity at excitatory synapses of GABA-IN and with the loss of correlation of NR2B-immunoreactivity at axo-spinous synapses. Conversely, we hypothesize that the lack of an ameliorative effect of the 3 mg/kg dose during ABA2 is likely to have been due, in part, to the failure of the mPFC of these animals to de-couple the mPFC synaptic circuitry linking axo-spinous synapses with suppression of food consumption and/or enhance NR2B expression at excitatory synapses on GABA-IN that suppress the excitatory out ow from mPFC that suppress food consumption (discussion is expanded in the Discussion section, with accompanying gure).
Correlations Between Wheel Activity and the Proportion of Excitatory Synapses Immunolabeled for NR2B Postsynaptically Earlier work from this lab showed that the mPFC pyramidal neurons projecting to dorsal medial striatum positively modulate wheel running of animals experiencing ABA while inhibition by GABAergic interneurons in mPFC suppresses wheel running (Santiago et al. 2021). We also reported earlier that both ketamine cohorts ran signi cantly less than the control group during ABA2 but not during ABA1 (Chen et al. 2018). These observations prompted us to assess correlations between wheel activity during ABA2 and the proportion of dendritic spines and shafts immunolabeled for postsynaptic NR2B at excitatory synapses in the mPFC of the two ketamine cohorts.
Correlations at axo-spinous excitatory synapses of pyramidal neurons. Figures 8a and 8b show the dayby-day values of R-values of Pearson correlation between wheel running during different time zones with NR2B immunoreactivity. Similar to the pattern observed for food consumption, there were clear differences in the correlation patterns between the two cohorts with regard to wheel activity. Note that wheels were absent during recovery phases, thereby precluding correlation analyses during the recovery days.The 30 mg/kg cohort exhibited similar dynamics of R-values across the different NR2B locations.
During FAA, the 30 mg/kg cohort, but not the 3mg/kg cohort, exhibited a dip of R-values for most NR2B locations except for the location at-PSD (Fig. 8b). This difference across the cohorts prompted us to look more closely at the correlations between wheel running and NR2B labeling during FAA (Fig. 9).
During the ABA2 FR period, both doses yielded signi cant reductions in averaged wheel running per day compared to the vehicle cohort (Chen et al. 2018). Although wheel activity between the two ketamine cohorts were not signi cantly different (mean ± SEM in units of km: 6.106 ± 1.69 for the 30 mg/kg cohort; 1.656 ± 1.74 for the 3 mg/kg cohort, p = 0.7938 by the unpaired t-test, Fig. 9a), correlation analysis revealed differences across the cohorts. Speci cally, at excitatory synapses of dendritic spines of pyramidal neurons of the 30 mg/kg cohort, a negative trend was found between wheel activity and the proportion of spines labeled for postsynaptic NR2B at all postsynaptic locations except for near-PSD (Pearson's R=-0.6274, p = 0.0959 for the 30 mg/kg cohort, Fig. 9b), indicating that those individuals with minimal FAA were those that had reduced NR2B expression at excitatory synapses of pyramidal neurons the most. No such trend was found in the 3 mg/kg cohort (Pearson's R = 0.3822, p = 0.3500 for the 3 mg/kg cohort, Fig. 9b).
Correlations at axo-shaft excitatory synapses of GABA-IN. Axo-shaft excitatory synapses of the 30 mg/kg cohort exhibited higher correlations between NR2B and wheel running than at axo-spinous synapses, especially during the dark hours of FR3 (21:00-7:00) (Fig. 8d, 9b). There was no signi cant group difference in the wheel activity between the two ketamine cohorts on ABA2 FR3's dark hours of 21:00-7:00 (mean ± SEM in units of km: 12.73 ± 2.18 for the 30 mg/kg cohort, 9.87 ± 2.46 for the 3 mg/kg cohort, p = 0.4086 by unpaired t-test, Fig. 9c). Yet, for the 30 mg/kg cohort, there was a highly signi cant positive correlation between wheel activity on ABA2 FR3 during dark phase 21:00-7:00 and the proportion of dendritic shafts of GABA-IN immunolabeled for postsynaptic NR2B at all postsynaptic locations other than near the PSD (Fig. 9d, Pearson's R = 0.9437, p = 0.0004 for the 30 mg/kg cohort). No such correlation was found in the 3 mg/kg cohort (Fig. 9d, Pearson's R = 0.1652, p = 0.6959 for the 3 mg/kg cohort).
Data reported were also analyzed by correlating wheel running and the levels of dendritic spines or shafts immunolabeled for NR2B. Correlation analyses counting every single NR2B immunolabel and normalizing the sum by the number of spines or shafts also revealed signi cance for the same sets of data.
Together, these data indicate that the dose of 30 mg/kg was more e cacious in promoting ABA resilience measured as reduced wheel running through gains of NR2B at axo-spinous excitatory synapses of pyramidal neurons and decreased NR2B-immunoreactivity at axo-shaft excitatory synapses of GABA-IN.
These observations are the opposite of the previous nding pertaining to the mPFC of animals without ketamine treatment (Santiago et al. 2021). The current data suggest that ketamine may mediate plasticity of the mPFC synaptic circuitry by unmasking mPFC pathways supporting adaptive behaviors, such as to suppress the hunger-evoked hyperactivity. Moreover, ketamine promoted synaptic changes in opposite directions depending on the cell type (pyramidal neuron vs GABA-IN), as was described above for the ketamine-induced changes supporting food consumption.

Correlations Between Body Weights and the Proportions of Excitatory Synapses Immunolabeled for NR2B
Body weight would be expected to be affected jointly by food consumption and wheel activity, each of which are regulated by speci c synaptic circuits within mPFC ( Pearson's correlation analyses revealed correlations between body weight and the levels of postsynaptic NR2B at excitatory synapses of dendritic spines, presumably of pyramidal neurons and dendritic shafts, presumably of GABA-IN. Similar to correlations between NR2B immunoreactivity and food consumption ( Fig. 6), correlation patterns of the two cohorts also differed for body weights (Fig. 10). R-values of the 3 mg/kg cohort were low during the two recovery phases and higher during FR periods. Also, the R-value dynamics of postsynaptic NR2B of the 3 mg/kg cohort were uniform across all synaptic locations (Fig. 10a). Oppositely, the 30 mg/kg cohort exhibited high R-values during the two recovery phases and lower R-values during FR periods. Within the 30 mg/kg cohort, postsynaptic NR2B near the PSD had a different R-value dynamic than NR2B at all other locations, as those near the PSD exhibited negative Rvalues during most of the course of ABA (Fig. 10b). Overall, ABA1 R3 and ABA2 R3 stood out as the days showing the strongest correlation between body weight and NR2B at excitatory synapses of pyramidal neurons' spines and GABA-IN's dendritic shafts. These correlations are shown in greater detail in Fig. 11.
Correlations at axo-spinous excitatory synapses on pyramidal neurons. Both the 3 mg/kg and 30 mg/kg cohorts responded to the single ketamine injection with increased body weight, relative to the vehicle group at recovery following ABA2, and only the 30mg/kg cohort improved in weight gain at recovery following ABA1 and during ABA2 FR period (Chen et al. 2018). However, two sample mean t-tests showed no differences between the body weights of the 3 mg/kg and 30 mg/kg ketamine cohorts on any of the days of recovery, including ABA1 R3 or ABA2 R3 (mean ± SEM in units of grams: 17.8 ± 0.619 for 30 mg/kg cohort, 18.3 ± 0.618 g for the 3 mg/kg cohort, p = 0.8199 on ABA1 R3, Fig. 11a; mean ± SEM in units of grams: 19.2 ± 0.507 for the 30 mg/kg cohort; 20.12 ± 0.580 for the 3 mg/kg cohort, p > 0.05 for ABA2 R3, Fig. 11c). In spite of the lack of difference in body weights between the two cohorts, comparisons of the correlations revealed differences across the doses. On ABA1 R3, a signi cant positive correlation was found between body weights and the proportion of spines immunolabeled for all postsynaptic NR2B other than near-PSD of the 30 mg/kg cohort but not the 3 mg/kg cohort (Pearson's R = 0.7667, p = 0.0443 for the 30 mg/kg cohort and Pearson's R = 0.0048, p = 0.9911 for the 3 mg/kg cohort, Fig. 11b). Similarly, on ABA2 R3, a positive correlation trend was found for the 30 mg/kg cohort but not the 3 mg/kg cohort (Pearson's R = 0.6316, p = 0.0930 for the 30 mg/kg cohort and Pearson's R = 0.3655, p = 0.3732, Fig. 11d). A positive correlation for the 30 mg/kg cohort (Fig. 11d) was still present when body weight was averaged from ABA1 R2-R7 (not shown), suggesting ultrastructural redistribution of NR2B that occurred and stabilized shortly after the end of ABA1's FR period.
Correlation analyses for the same days and ultrastructural location of NR2B revealed equally signi cant outcomes using NR2B levels at excitatory synapses instead of the proportions of NR2B-immunoreactive excitatory synapses.  Fig. 11f). Oppositely, a positive trend was found in the 30 mg/kg cohort (Pearson's R = 0.5946, p = 0.1200 for the 30 mg/kg cohort, Fig. 11f).
The same behavioral data set was correlated to NR2B levels, assessed by counting every single NR2B immunolabel and normalizing the sum by the number of dendritic shafts encountered. The outcome revealed greater signi cances of correlations (Fig. 11g, h). In contrast to the negative trend shown in Fig. 11e for the proportions of synapses immunolabeled, there was a signi cantly negative correlation between body weight and the level of dendritic shafts immunolabeled for NR2B for the 3mg/kg cohort on ABA1 R3, and no correlation or trend for the 30mg/kg cohort (Pearson's R = 0.3562, p = 0.4330 for the 30mg/kg cohort and Pearson's R=-0.7176, p = 0.0450 for the 3mg/kg cohort, Fig. 11g). Likewise, in contrast to the negative trend shown in Fig. 11f, there was a signi cant positive correlation between body weight and the level of dendritic shafts immunolabeled for NR2B for the 30mg/kg cohort but only a negative trend for the 3mg/kg cohort (Pearson's R = 0.7428, p = 0.0348 for the 30mg/kg cohort and Pearson's R=-0.6738, p = 0.0670 for the 3mg/kg cohort, Fig. 11h).
Overall, at dendritic spines of pyramidal neurons, the increase in the proportion and levels of NR2B had an ameliorative effect on body weight restoration and more so for the 30 mg/kg dose than for the 3 mg/kg dose (Fig. 11b). In sharp contrast to the changes at dendritic spines, the changes at excitatory synapses on GABA-IN re ected losses of NR2B of 3 mg/kg cohort, seen as ameliorative for weight retention, changing to a positive correlation by ABA2 and enhanced NR2B that contributed to 30 mg/kg cohort's body weight gain.

Discussion
Ketamine is an open channel NMDAR blocker. As such, ketamine that was injected while animals exhibited food restriction-evoked maladaptive hyperactivity and suppressed food consumption may have blocked excitatory synapses within circuits that were mediating those maladaptive behaviors. We hypothesize that ketamine's blockade of those synapses within mPFC circuitry subserving maladaptive behaviors may have enabled the competing adaptive behaviors' synaptic circuits to become more dominant, thereby enabling a switch of animals' behavior from the maladaptive to more adaptive ones. In the following sections, we consider the types of mPFC circuitry and directions of NR2B changes at synapses that would support this hypothesis.

Food Consumption During Recovery Following Ketamine Treatment
The contribution by pyramidal neurons' postsynaptic NR2B in enhancing food consumption Behaviorally, we observed that 30 mg/kg ketamine protected mice against ABA vulnerability by increasing food consumption during ABA and during the recovery phase following both ABA1 and ABA2. During recovery from ABA1, there was a positive trend for the 30 mg/kg dose and not for the 3 mg/kg dose between increased food consumption and NR2B immunoreactivity at excitatory synapses on pyramidal neurons. This trend suggests that the more e cacious dose of 30 mg/kg following ABA1 may have been due, in part, to an increase in the proportion of dendritic spines of pyramidal neurons immunolabeled for NR2B. The positive correlation was observed almost everywhere (at PSD, extrasynaptically, in the synaptic cleft, and cytoplasmically) (Fig. 7b). This ts with a previous nding indicating that an increase of the E/I (excitatiion to inhibition) ratio of synaptic inputs to mPFC pyramidal neurons which are excitatory themselves and project to multiple subcortical regions promotes food consumption (Du et al. 2022  Following ABA2, the e cacious dose of 30 mg/kg correlated not at all with NR2B levels at excitatory synapses onto pyramidal neurons in mPFC (Fig. 7d). This could be due to the disengagement of the mPFC→DR GABA pathway as the major contributor to the enhanced food consumption. More likely, the improved food consumption is due to a counterbalanced weakening of another mPFC excitatory out ow that suppresses food consumption (Circuit A after Ketamine in Fig. 12), such as the mPFC pyramidal neurons that directly target serotonergic neurons in DR (mPFC◊DR 5HT ) (Nectow et al. 2017;Jankowski and Sesack 2004). In the PFC, there are multiple populations of pyramidal neurons that regulate food consumption. Besides those projecting to DR, mPFC neurons that project to lateral hypothalamus (LH) promote cued food consumption of non-starving animals (Holland and Petrovich 2005) but can also suppress feeding even of food-deprived animals, if it targets the glutamatergic neurons in LH (mPFC→LH glut ) (Jennings et al. 2013). Another excitatory out ow from the mPFC to the lateral habenula (mPFC→LHb) is theorized to underlie stress-evoked anxiety and anhedonia in humans (Vadovicova 2014). The excitatory out ow from mPFC suppressing either of these food consumption-suppressing subcortical centers may have been weakened by the 30 mg/kg dose of ketamine in time for recovery from ABA2, thereby diminishing the positive correlation trend seen after ABA1. Published work already exists indicating that the prophylactic stress-buffering effects of ketamine involve synaptic plasticity of the mPFC→DR that targets the 5-HT neurons in DR (Dolzani et al. 2018).
The idea that there are parallel antagonistic pathways from mPFC for food consumption regulation is supported by considering the outcome of treating ABA animals with the ine cacious dose of 3 mg/kg.
The outcome was that food consumption correlated negatively and strongly with NR2B prevalence at excitatory synapses formed on pyramidal neurons. This may re ect the persistent dominant in uence by the food consumption-suppressive excitatory out ow from mPFC (mPFC→LH glut ; mPFC→LHb; mPFC◊DR 5HT ) described above (Circuit A in Fig. 12), when the synaptic circuitry underlying the maladaptive behavior is not blocked su ciently by ketamine. Further studies of the synaptic circuitry within DR and mPFC would be necessary to understand more fully about the mechanism of action of the e cacious dose of ketamine.
The contribution of GABA-IN's postsynaptic NR2B in the enhanced food consumption. Our earlier study that did not employ ketamine treatment showed that chemogenetic stimulation of the mPFC→DR pathway (Circuit B in Fig. 12) could not override the strong GABAergic inhibition within mPFC that inhibited feeding (Du et al. 2022). The e cacious dose of ketamine may have blocked the active synapses underlying maladaptive behavior (Circuit A after Ketamine in Fig. 12). This ketamine-mediated blockade of the excitatory synapses on GABA-IN in Layer 5 (Circuit A after Ketamine, Fig. 12) may have enabled Circuit B's excitatory out ow from the mPFC to dominate behavior as ketamine's acute mechanism in addition to the changes hypothesized above regarding NR2B expression at excitatory synapses on GABA-IN in Layer 1 to support the adaptive changes that began during recovery from ABA1 ( Fig. 7e) and lasted for more than 20 days (see Fig. 7f).
In the absence of ketamine, the relative prevalence of GABAergic innervation of Layer 5 pyramidal neurons forming the mPFC→DR pathway correlated negatively with food consumption (Du et al. 2022). We had learned in an earlier chemogenetic interrogation experiment that mPFC contains at least two parallel antagonistic pathways regulating food restriction-evoked hyperactivity of ABA animals: those that promote wheel running, which project to dorsal medial striatum (mPFC→DMS) and those that project elsewhere and suppress wheel running. Furthermore, we had learned that GABA-IN of the mPFC that are recruited by the chemogenetic stimulation of mPFC→DMS pyramidal neurons mostly innervate cell bodies of those pyramidal neurons that suppress running (Circuit B in Fig. 13) and not the cell bodies of pyramidal neurons projecting to DMS (Circuit A in Fig. 13) (Santiago et al. 2021). A change in circuit consistent with the gain of resilience after ketamine treatment is that 30 mg/kg of ketamine blocks the active synapses, and most critically those excitatory synapses on PV + neurons inhibiting the adaptive circuit, enabling the activity of the pyramidal neurons supporting the suppression of running to dominate behavior (Circuit B after Ketamine).
During ABA2, the most prominent correlation of NR2B levels to wheel activity appeared at Layer 1a excitatory synapses on GABA-IN of the 30 mg/kg cohort. As stated above when discussing the circuits underlying increases in food consumption, the most prevalent type of GABA-IN in Layer 1a are the NDNF + GABA-IN that mediate disinhibition of pyramidal neurons through inhibition of PV + GABA-IN. Here, the correlation was strongly positive (Fig. 9d), indicating that those individuals with the weakest NDNF + GABAergic inhibition, due to weakened excitatory synaptic input, minimized the level of wheel running the most, i.e., gained ABA resilience. This correlation is consistent with the idea that 30 mg/kg of ketamine blocked, then eliminated NR2B expression at excitatory synapses on NDNF + GABA-IN, thereby reducing the disinhibition of the maladaptive Circuit A (Circuit A after Ketamine in Fig. 13). The strong correlation indicates that this reduction of disinhibition in Layer 1a upon the maladaptive circuit was most in uential for reducing the food restriction-evoked hyperactivity. While the NDNF + GABA-IN in Layer 1a could be recruited following activation of Circuit A pyramidal neurons, it is likely that the PV + GABA-IN targeted by NDNF + GABA-IN are distinct from the population of PV + GABA-IN recruited through activity of Circuit A's pyramidal neurons (PV1 cells of Fig. 13, left panel). This is because the PV + cells inhibited by NDNF+ (PV2 cells in the left panel) would have to be those that remained relatively inactive while animals exhibited maladaptive behaviors driven by Circuit A, thereby escaping ketamine's activity-dependent blockade and becoming active after the ketamine treatment (PV2 cells, in the right panel).
Pyramidal neurons' secondary contribution to the ketamine-mediated suppression of wheel running. The 30 mg/kg cohort also exhibited a weak negative trend between excitatory synaptic drive of pyramidal neurons and wheel activity (Fig. 9b). This is consistent with the idea that the excitatory synaptic drive of pyramidal neurons that suppress running was enhanced through redistribution of NR2B-NMDARs towards excitatory synapses on pyramidal neurons of the adaptive circuit that had been dormant prior to ketamine treatment (Circuit B after Ketamine, Fig. 13), while excitatory synapses on pyramidal neurons of the maladaptive circuit that had been active during ABA1 became blocked by ketamine (Circuit A after Ketamine), culminating in the slight reduction of NR2B at pyramidal neurons, overall (5 lled stars associated with pyramidal neurons in the left panel to 3 lled starts in right panel).

Body Weight After Ketamine Injection
Contribution by postsynaptic NR2B of pyramidal neurons and GABA-IN in body weight restoration associated with the 30 mg/kg cohort Body weight is regulated jointly by wheel running and food consumption. However, with regard to ABA1, we observed a signi cant ameliorative effect of the 30 mg/kg dose of ketamine on body weight restoration during recovery, when the running wheel was no longer accessible to mice (Chen et al. 2018), suggesting that the pathway underlying body weight restoration could have been related to those pathways regulating food consumption more than of wheel running. The prominently positive correlation observed between NR2B levels at pyramidal neurons' excitatory synapses and body weight restoration following ABA1 (Fig. 11b) suggests that this was contributed largely by the enhanced excitability of the mPFC→DR GABA pathway (Du et al. 2022;Nectow et al. 2017) (Adaptive Circuit B, right panel in Fig. 12).
The loss of correlation of NR2B levels at excitatory synapses on GABA-IN for the 30 mg/kg cohort, together with the persistently negative correlations for the cohort that received the ineffective 3 mg/kg dose (Fig. 11e, f, g, h) indicates that the relative di culty with body weight restoration of the 3 mg/kg ketamine cohort was due to the persistently strong activity of excitatory synapses on Layer 1 GABA-IN that disinhibited the maladaptive behavior (circuit A, left panel), which could be blocked effectively by 30 mg/kg of ketamine (Fig. 12, right).
During ABA2 and recovery from it, the positive correlation between NR2B levels at excitatory synapses on pyramidal neurons and body weight weakened slightly (Fig. 11d), relative to the recovery from ABA1 (Fig. 11b). This suggests that the ameliorative behavior leading to better body weight restoration by the 30 mg/kg dose following ABA1, compared to the 3 mg/kg dose (Chen et al. 2018) is likely to re ect the strong positive correlation of NR2B levels at pyramidal neurons of the adaptive pathway (mPFC→DR GABA , Circuit B in Fig. 12) which could be revealed due to ketamine blockade, then removal of NR2B from pyramidal neurons of the maladaptive pathway, leading to weakening of the maladaptive pathway (mPFC→LH glut , mPFC→LHb; mPFC◊DR 5HT ) (Circuit A after Ketamine in Fig. 12).
Added to the plasticity at pyramidal neurons is the strengthened suppression of the maladaptive pathway mediated by GABA-IN, re ected by the signi cantly positive correlation between NR2B levels at excitatory synapses of GABA-IN and better body weight restoration (Figs. 11h). As hypothesized above for the action of the effective dose of ketamine for food consumption, we believe the effective dose of ketamine successfully blocked the maladaptive food consumption pathways, such as the excitatory afferents to Layer 1 GABA-IN that disinhibited the maladaptive pathways (circuit A after Ketamine, Fig. 12), thereby enabling up-regulation of NR2B levels at excitatory synaptic sites on GABA-IN that disinhibit adaptive pathways (Circuit B after Ketamine, Fig. 12), such as the mPFC◊DR GABA .

Conclusion
The site of action of ketamine is likely to be distributed across multiple brain regions, although the literature pertaining to ketamine's mode of action is dominated by research examining rather selective brain regions -mPFC, hippocampus and LHb. To our knowledge, this is one of only two studies examining ketamine's effect on synaptic properties as it relates to ABA, an animal model of AN.
Our study is also unique in having examined the impact of ketamine administered during midadolescence, while most other studies examined the impact of ketamine upon psychiatric diseases and animal models in adulthood. Adolescence is when multiple mental illnesses arise (Whitford et al. 2007), and anorexia nervosa (AN) is one such mental illness that emerges most prominently during adolescence . This is the reason we chose mid-adolescent female mice to be the subjects of our study. Brain maturation and development, including pruning, are very active during adolescence in mPFC, especially in response to stress (Whitford et al. 2007; Juraska and Willing 2017; Anderson 2022; Delevich et al. 2021). The active turnover of synapses during mid-adolescence may have enhanced the receptivity of mPFC to ketamine more than in adulthood, since administration of the same ketamine treatment to animals undergoing ABA during late adolescence/early adulthood was not e cacious (Aoki 2020). We are currently exploring alternative e cacious doses of ketamine for ameliorating ABA vulnerability in adulthood.
Our overall nding of this initial study is that the correlations between NR2B-NMDAR levels at synapses in layer 1a of mPFC and variables related to ABA vulnerability (wheel activity, food consumption, body restoration) require the consideration of plasticity at multiple excitatory corticofugal pathways that work antagonistically. For example, we had learned from earlier chemogenetic studies (Santiago et al. 2021) that there must be two or more pathways of mPFC origin subserving enhancement versus suppression of food restriction-evoked hyperactivity. The current ndings enabled us to recognize that the effective dose of ketamine ameliorated ABA vulnerability by suppressing certain pathways while boosting others. By incorporating the knowledge from prior studies that interrogated the mPFC excitatory out ows chemogenetically, we could surmise that the suppressed pathways might include the mPFC◊DMS (Fig. 13) but are likely to include additional parallel pathways that suppress food intake (Fig. 12).
Conversely, we hypothesize that pathways enhanced in synaptic strength through increased levels of NR2B might include the mPFC◊DR GABA pathway (Fig. 12) but is also likely to include mPFC corticofugal pathways that suppress wheel running (Fig. 13). Moreover, we learned that NR2B expression at excitatory synapses onto GABA-IN exhibit plasticity that supports resilience most consistently (strong correlations), yielding both reduced wheel running ( Fig. 9d and Fig. 13), increased food consumption (Figs. 7e and 7f; Fig. 12) and increased body weight (Figs. 11g and 11h; Fig. 12).
The multi-directional and multiple pathways that are differentially modulated across the e cacious versus ine cacious doses t with the idea hypothesized at the onset -namely that 30 mg/kg of ketamine, more so than the 3 mg/kg dose, blocks the active pathways subserving maladaptive behaviors, thereby enabling the relatively quiet pathways to emerge as dominant players supporting adaptive behavior. Future studies that extend the analysis to additional brain regions promise to provide a more complete understanding of the mechanism of action of ketamine. Moreover, analysis of NR2B redistribution evoked by sub-anesthetic doses of ketamine at excitatory synapses of de nitively identi ed corticofugal pathways would greatly facilitate our knowledge of the multiple parallel pathways of the mPFC that coordinate the switching of maladaptive behaviors to adaptive behaviors.

Declarations
Data availability: All data not included in the manuscript can be made available upon request.

Figure 1
The activity-based anorexia (ABA) animal model Panel a. The ABA timeline Acclimation to wheels started on postnatal day 36 (P36) and lasted for 5 days. The rst day of food restriction (ABA1 FR1) started on P41 and lasted for 3 days. On ABA1 FR2, which is the second day of ABA1's food restriction phase (FR), all animals received either 3mg/kg or 30mg/kg of ketamine by intraperitoneal injection. The recovery phase after ABA1 FR started on P44 and lasted for 7 days. Animals were then re-acclimated to wheels for 4 days, starting on P51. Animals were food restricted again (ABA2 FR) for 4 days, starting on P55. The second recovery phase, which followed ABA2 FR, started on P59 and lasted for 5 days.
Panel b. Schematic of the 24-hr schedule during FR days FR started at 1pm on P41 for ABA1 and on P55 for ABA2. The 2-hour food access period of each FR day started at 7pm and ended at 9pm. Lights were turned on from 7am to 7pm everyday (light phase). Lights were off from 7pm to 7am (dark phase). 3mg/kg or 30mg/kg ketamine were injected at 6pm of ABA1 FR2, 1 hr before feeding. FAA (food anticipatory activity) period refers to the hours of 1 pm to 7 pm, when hunger evokes hyperactivity of ABA animals.  PEG labeling re ective of NR2B immunoreactivity is located at the cleft (gold particle 1, blue arrow) and at the PSD (gold particle 2, blue arrow). pre = presynaptic. Panel c shows an example of an excitatory synapse on a dendritic shaft, based on the presence of a mitochondrial pro le on the postsynaptic side (m) and PSD. One PEG labeling is at the PSD (blue arrow).
Another is cytoplasmic (pink arrow). This dendritic shaft is presumed to belong to a GABA-IN (White & Keller, A. 1989). NR2B immunolabels appearing at locations highlighted with blue arrowheads are presumed to be functional since they were on the plasma membrane, while those at locations highlighted with pink arrowheads are presumed to be of reserve or recycling pool of NR2B.
Calibration bar = 500 nm and applies to both panels b and c.     The ameliorative wheel activity effect of the 30 mg/kg ketamine during ABA2 was associated with decreased NR2B at excitatory synapses on GABA-IN Panels a and c: There was no signi cant difference between wheel activities of the two cohorts on ABA2 FR4 FAA or ABA2 FR3 dark 21-7.
Panel b: At excitatory synapses of pyramidal neurons' dendritic spines, there was a negative trend between wheel activity during FAA of ABA2 FR4 and the proportion of postsynaptic NR2B at all locations except near PSD for the 30mg/kg cohort and no correlation for the 3mg/kg cohort.

Figure 11
Improvement of body weight retention among the 30 mg/kg cohort was contributed by increased NR2B at excitatory synapses on pyramidal neurons, together with decreased NR2B at excitatory synapses on GABA-IN during recovery from ABA1 and by the increased NR2B at excitatory synapses on GABA-IN during recovery from ABA2. Panels a and b: On both ABA1 R3 and ABA2 R3, there was no signi cant body weight difference. between the two cohorts.   NR2B activity at synapses of parallel circuits within the mPFC regulating food restriction-evoked wheel running Panel a: Circuits prior ketamine treatment Panel b: Circuits following ketamine injection. With the e cacious dose of ketamine (30 mg/kg), blockade of the more active synapses in Circuit A that mediate maladaptive behavior leads to the emergence of the more adaptive behavior mediated by Circuit B. Circuit A's activity is also suppressed due to decreased expression of NR2B at GABA-IN in Layer 1 (Fig 9d). PV1 is recruited by pyramidal neurons (pyr) mediating the maladaptive circuit A, while PV2 is not. See Fig 12 for additional gure legends.